Mixed responses to targeted therapy driven by chromosomal instability through p53 dysfunction and genome doubling.

The phenomenon of mixed/heterogenous treatment responses to cancer therapies within an individual patient presents a challenging clinical scenario. Furthermore, the molecular basis of mixed intra-patient tumor responses remains unclear. Here, we show that patients with metastatic lung adenocarcinoma harbouring co-mutations of EGFR and TP53, are more likely to have mixed intra-patient tumor responses to EGFR tyrosine kinase inhibition (TKI), compared to those with an EGFR mutation alone. The combined presence of whole genome doubling (WGD) and TP53 co-mutations leads to increased genome instability and genomic copy number aberrations in genes implicated in EGFR TKI resistance. Using mouse models and an in vitro isogenic p53-mutant model system, we provide evidence that WGD provides diverse routes to drug resistance by increasing the probability of acquiring copy-number gains or losses relative to non-WGD cells. These data provide a molecular basis for mixed tumor responses to targeted therapy, within an individual patient, with implications for therapeutic strategies.

Targeting of vulnerabilities of drug-tolerant persisters identified through functional genetics delays tumor relapse.

Drug-tolerant persisters (DTPs) are a rare subpopulation of cells within a tumor that can survive therapy through nongenetic adaptive mechanisms to develop relapse and repopulate the tumor following drug withdrawal. Using a cancer cell line with an engineered suicide switch to kill proliferating cells, we perform both genetic screens and compound screens to identify the inhibition of bromodomain and extraterminal domain (BET) proteins as a selective vulnerability of DTPs. BET inhibitors are especially detrimental to DTPs that have reentered the cell cycle (DTEPs) in a broad spectrum of cancer types. Mechanistically, BET inhibition induces lethal levels of ROS through the suppression of redox-regulating genes highly expressed in DTPs, including GPX2, ALDH3A1, and MGST1. In vivo BET inhibitor treatment delays tumor relapse in both melanoma and lung cancer. Our study suggests that combining standard of care therapy with BET inhibitors to eliminate residual persister cells is a promising therapeutic strategy.

APOBEC3 as a driver of genetic intratumor heterogeneity.

Our recent study revealed that APOBEC3B is upregulated during the preinvasive stages of non-small cell lung cancer and breast cancer. In addition to its role in mediating single nucleotide variants, we propose that APOBEC3 promotes copy number intratumor heterogeneity prior to invasion, providing a substrate for cancer evolution.

Investigating chromosomal instability in long-term survivors with glioblastoma and grade 4 astrocytoma.

Background: Only a small group of patients with glioblastoma multiforme (GBM) survives more than 36 months, so-called long-term survivors. Recent studies have shown that chromosomal instability (CIN) plays a prognostic and predictive role among different cancer types. Here, we compared histological (chromosome missegregation) and bioinformatic metrics (CIN signatures) of CIN in tumors of GBM typical survivors (≤36 months overall survival), GBM long-term survivors and isocitrate dehydrogenase (IDH)-mutant grade 4 astrocytomas. Methods: Tumor sections of all gliomas were examined for anaphases and chromosome missegregation. Further CIN signature activity analysis in the The Cancer Genome Atlas (TCGA)-GBM cohort was performed. Results: Our data show that chromosome missegregation is pervasive in high grade gliomas and is not different between the 3 groups. We find only limited evidence of altered CIN levels in tumors of GBM long-term survivors relative to the other groups, since a significant depletion in CIN signature 11 relative to GBM typical survivors was the only alteration detected. In contrast, within IDH-mutant grade 4 astrocytomas we detected a significant enrichment of CIN signature 5 and 10 activities and a depletion of CIN signature 1 activity relative to tumors of GBM typical survivors. Conclusions: Our data suggest that CIN is pervasive in high grade gliomas, however this is unlikely to be a major contributor to the phenomenon of long-term survivorship in GBM. Nevertheless, further evaluation of specific types of CIN (signatures) could have prognostic value in patients suffering from grade 4 gliomas.

Deciphering the mechanism of Tinospora cordifolia extract on Th17 cells through in-depth transcriptomic profiling and in silico analysis.

Naive CD4+ T cells differentiate into effector (Th1, Th2, Th17) cells and immunosuppressive (Treg) cells upon antigenic stimulation in the presence of a specific cytokine milieu. The T cell in vitro culture system provides a very efficient model to study compounds' therapeutic activity and mechanism of action. Tinospora cordifolia (Willd.) Hook.f. & Thomson (Family. Menispermaceae) is one of the widely used drugs in Ayurveda (ancient Indian system of medicine) for various ailments such as inflammatory conditions, autoimmune disorders, and cancer as well as for promoting general health. In vitro and in vivo studies on immune cells comprising dendritic cells, macrophages, and B cells suggest its immune-modulating abilities. However, to date, the effect of T. cordifolia on individual purified and polarized T cell subsets has not been studied. Studying drug effects on T cell subsets is needed to understand their immunomodulatory mechanism and to develop treatments for diseases linked with T cell abnormalities. In this study, we examined the immunomodulatory activity of T. cordifolia on primary CD4+ T cells, i.e., Th1, Th17, and iTreg cells. An aqueous extract of T. cordifolia was non-cytotoxic at concentrations below 1500 µg/ml and moderately inhibited the proliferation of naive CD4+ T cells stimulated with anti-CD3ε and anti-CD28 for 96 h. T. cordifolia treatment of naive CD4+ T cells differentiated under Th17-polarizing conditions exhibited reduced frequency of IL-17 producing cells with inhibition of differentiation and proliferation. For the first time, in-depth genome-wide expression profiling of T. cordifolia treated naive CD4+ T cells, polarized to Th17 cells, suggests the broad-spectrum activity of T. cordifolia. It shows inhibition of the cytokine-receptor signaling pathway, majorly via the JAK-STAT signaling pathway, subsequently causing inhibition of Th17 cell differentiation, proliferation, and effector function. Additionally, the molecular docking studies of the 69 metabolites of T. cordifolia further substantiate the inhibitory activity of T. cordifolia via the cytokine-receptor signaling pathway. Furthermore, in vitro polarized Th1 and iTreg cells treated with T. cordifolia extract also showed reduced IFN-γ production and FoxP3 expression, respectively. This study provides insight into the plausible mechanism/s of anti-inflammatory activity of T. cordifolia involving T cells, mainly effective in Th17-associated autoimmune and inflammatory diseases.

Induction of APOBEC3 Exacerbates DNA Replication Stress and Chromosomal Instability in Early Breast and Lung Cancer Evolution.

APOBEC3 enzymes are cytosine deaminases implicated in cancer. Precisely when APOBEC3 expression is induced during cancer development remains to be defined. Here we show that specific APOBEC3 genes are upregulated in breast ductal carcinoma in situ, and in preinvasive lung cancer lesions coincident with cellular proliferation. We observe evidence of APOBEC3-mediated subclonal mutagenesis propagated from TRACERx preinvasive to invasive non-small cell lung cancer (NSCLC) lesions. We find that APOBEC3B exacerbates DNA replication stress and chromosomal instability through incomplete replication of genomic DNA, manifested by accumulation of mitotic ultrafine bridges and 53BP1 nuclear bodies in the G1 phase of the cell cycle. Analysis of TRACERx NSCLC clinical samples and mouse lung cancer models revealed APOBEC3B expression driving replication stress and chromosome missegregation. We propose that APOBEC3 is functionally implicated in the onset of chromosomal instability and somatic mutational heterogeneity in preinvasive disease, providing fuel for selection early in cancer evolution. SIGNIFICANCE: This study reveals the dynamics and drivers of APOBEC3 gene expression in preinvasive disease and the exacerbation of cellular diversity by APOBEC3B through DNA replication stress to promote chromosomal instability early in cancer evolution.This article is highlighted in the In This Issue feature, p. 2355.

Insights from a Pan India Sero-Epidemiological survey (Phenome-India Cohort) for SARS-CoV2.

To understand the spread of SARS-CoV2, in August and September 2020, the Council of Scientific and Industrial Research (India) conducted a serosurvey across its constituent laboratories and centers across India. Of 10,427 volunteers, 1058 (10.14%) tested positive for SARS-CoV2 anti-nucleocapsid (anti-NC) antibodies, 95% of which had surrogate neutralization activity. Three-fourth of these recalled no symptoms. Repeat serology tests at 3 (n = 607) and 6 (n = 175) months showed stable anti-NC antibodies but declining neutralization activity. Local seropositivity was higher in densely populated cities and was inversely correlated with a 30-day change in regional test positivity rates (TPRs). Regional seropositivity above 10% was associated with declining TPR. Personal factors associated with higher odds of seropositivity were high-exposure work (odds ratio, 95% confidence interval, p value: 2.23, 1.92-2.59, <0.0001), use of public transport (1.79, 1.43-2.24, <0.0001), not smoking (1.52, 1.16-1.99, 0.0257), non-vegetarian diet (1.67, 1.41-1.99, <0.0001), and B blood group (1.36, 1.15-1.61, 0.001).

Chromosomal instability drives metastasis through a cytosolic DNA response.

Chromosomal instability is a hallmark of cancer that results from ongoing errors in chromosome segregation during mitosis. Although chromosomal instability is a major driver of tumour evolution, its role in metastasis has not been established. Here we show that chromosomal instability promotes metastasis by sustaining a tumour cell-autonomous response to cytosolic DNA. Errors in chromosome segregation create a preponderance of micronuclei whose rupture spills genomic DNA into the cytosol. This leads to the activation of the cGAS-STING (cyclic GMP-AMP synthase-stimulator of interferon genes) cytosolic DNA-sensing pathway and downstream noncanonical NF-κB signalling. Genetic suppression of chromosomal instability markedly delays metastasis even in highly aneuploid tumour models, whereas continuous chromosome segregation errors promote cellular invasion and metastasis in a STING-dependent manner. By subverting lethal epithelial responses to cytosolic DNA, chromosomally unstable tumour cells co-opt chronic activation of innate immune pathways to spread to distant organs.

Treatment-Induced Mutagenesis and Selective Pressures Sculpt Cancer Evolution.

Despite the great progress in our understanding of the molecular basis of human cancer, the heterogeneity of individual tumors and the evolutionary pressures imposed by therapy have hampered our ability to effectively eradicate and control this disease. How, therefore, do cancers evolve under the selective pressures of cancer therapy? Recent studies have linked both primary (or de novo) and acquired treatment resistance to intratumor heterogeneity and clonal evolution. Resistance to targeted therapies often includes mutation of the drug target itself and aberrations of pathways upstream of, downstream from, or parallel to the drug target. For systemic chemotherapies, discrete and recurrent resistance-conferring genetic aberrations have eluded the community, due in part to their wide-ranging mutagenic effects. In this review, we discuss different patterns of clonal evolution during treatment-specific selective pressures and focus on the genetic mechanisms of treatment resistance that have emerged to both targeted therapies and chemotherapies.

Constraints in cancer evolution.

Next-generation deep genome sequencing has only recently allowed us to quantitatively dissect the extent of heterogeneity within a tumour, resolving patterns of cancer evolution. Intratumour heterogeneity and natural selection contribute to resistance to anticancer therapies in the advanced setting. Recent evidence has also revealed that cancer evolution might be constrained. In this review, we discuss the origins of intratumour heterogeneity and subsequently focus on constraints imposed upon cancer evolution. The presence of (1) parallel evolution, (2) convergent evolution and (3) the biological impact of acquiring mutations in specific orders suggest that cancer evolution may be exploitable. These constraints on cancer evolution may help us identify cancer evolutionary rule books, which could eventually inform both diagnostic and therapeutic approaches to improve survival outcomes.

Evolutionary dynamics in pre-invasive neoplasia.

Mutational processes occur in normal tissues from conception throughout life. Field cancerization describes the preconditioning of an area of epithelium to tumor growth. Pre-invasive lesions may arise in these fields, however only a minority of pre-invasive neoplasia progresses to overt malignancy. Within this review we discuss recent advances in our understanding of genomic instability processes in normal tissue, describe evolutionary dynamics in pre-invasive disease and highlight current evidence describing how increasing genomic instability may drive the transition from pre-invasive to invasive disease. Appreciation of the evolutionary rulebooks that operate in pre-invasive neoplasia may facilitate screening strategies, risk-stratification of pre-invasive lesions and precipitate novel preventative treatments in at-risk patient populations.

DNA replication stress mediates APOBEC3 family mutagenesis in breast cancer.

BACKGROUND: The APOBEC3 family of cytidine deaminases mutate the cancer genome in a range of cancer types. Although many studies have documented the downstream effects of APOBEC3 activity through next-generation sequencing, less is known about their upstream regulation. In this study, we sought to identify a molecular basis for APOBEC3 expression and activation. RESULTS: HER2 amplification and PTEN loss promote DNA replication stress and APOBEC3B activity in vitro and correlate with APOBEC3 mutagenesis in vivo. HER2-enriched breast carcinomas display evidence of elevated levels of replication stress-associated DNA damage in vivo. Chemical and cytotoxic induction of replication stress, through aphidicolin, gemcitabine, camptothecin or hydroxyurea exposure, activates transcription of APOBEC3B via an ATR/Chk1-dependent pathway in vitro. APOBEC3B activation can be attenuated through repression of oncogenic signalling, small molecule inhibition of receptor tyrosine kinase signalling and alleviation of replication stress through nucleoside supplementation. CONCLUSION: These data link oncogene, loss of tumour suppressor gene and drug-induced replication stress with APOBEC3B activity, providing new insights into how cytidine deaminase-induced mutagenesis might be activated in tumourigenesis and limited therapeutically.

TP53 mutated glioblastoma stem-like cell cultures are sensitive to dual mTORC1/2 inhibition while resistance in TP53 wild type cultures can be overcome by combined inhibition of mTORC1/2 and Bcl-2.

BACKGROUND: Glioblastoma is the most malignant tumor of the central nervous system and still lacks effective treatment. This study explores mutational biomarkers of 11 drugs targeting either the RTK/Ras/PI3K, the p53 or the Rb pathway using 25 patient-derived glioblastoma stem-like cell cultures (GSCs). RESULTS: We found that TP53 mutated GSCs were approximately 3.5 fold more sensitive to dual inhibition of mammalian target of rapamycin complex 1 and 2 (mTORC1/2) compared to wild type GSCs. We identified that Bcl-2(Thr56/Ser70) phosphorylation contributed to the resistance of TP53 wild type GSCs against dual mTORC1/2 inhibition. The Bcl-2 inhibitor ABT-263 (navitoclax) increased sensitivity to the mTORC1/2 inhibitor AZD8055 in TP53 wild type GSCs, while sensitivity to AZD8055 in TP53 mutated GSCs remained unchanged. CONCLUSION: Our data suggest that Bcl-2 confers resistance to mTORC1/2 inhibitors in TP53 wild type GSCs and that combined inhibition of both mTORC1/2 and Bcl-2 is worthwhile to explore further in TP53 wild type glioblastomas, whereas in TP53 mutated glioblastomas dual mTORC1/2 inhibitors should be explored.

Tumor Evolutionary Principles: How Intratumor Heterogeneity Influences Cancer Treatment and Outcome.

Recent studies have shown that intratumor heterogeneity contributes to drug resistance in advanced disease. Intratumor heterogeneity may foster the selection of a resistant subclone, sometimes detectable prior to treatment. Next-generation sequencing is enabling the phylogenetic reconstruction of a cancer's life history and has revealed different modes of cancer evolution. These studies have shown that cancer evolution is not always stochastic and has certain constraints. Consideration of cancer evolution may enable the better design of clinical trials and cancer therapeutics. In this review, we summarize the different modes of cancer evolution and how this might impact clinical outcomes. Furthermore, we will discuss several therapeutic strategies for managing emergent intratumor heterogeneity.

Genetic biomarkers of drug response for small-molecule therapeutics targeting the RTK/Ras/PI3K, p53 or Rb pathway in glioblastoma.

Glioblastoma is the most deadly and frequently occurring primary malignant tumor of the central nervous system. Genomic studies have shown that mutated oncogenes and tumor suppressor genes in glioblastoma mainly occur in three pathways: the RTK/Ras/PI3K signaling, the p53 and the Rb pathways. In this review, we summarize the modulatory effects of genetic aberrations in these three pathways to drugs targeting these specific pathways. We also provide an overview of the preclinical efforts made to identify genetic biomarkers of response and resistance. Knowledge of biomarkers will finally promote patient stratification in clinical trials, a prerequisite for trial design in the era of precision medicine.

AntiAngioPred: A Server for Prediction of Anti-Angiogenic Peptides.

The process of angiogenesis is a vital step towards the formation of malignant tumors. Anti-angiogenic peptides are therefore promising candidates in the treatment of cancer. In this study, we have collected anti-angiogenic peptides from the literature and analyzed the residue preference in these peptides. Residues like Cys, Pro, Ser, Arg, Trp, Thr and Gly are preferred while Ala, Asp, Ile, Leu, Val and Phe are not preferred in these peptides. There is a positional preference of Ser, Pro, Trp and Cys in the N terminal region and Cys, Gly and Arg in the C terminal region of anti-angiogenic peptides. Motif analysis suggests the motifs "CG-G", "TC", "SC", "SP-S", etc., which are highly prominent in anti-angiogenic peptides. Based on the primary analysis, we developed prediction models using different machine learning based methods. The maximum accuracy and MCC for amino acid composition based model is 80.9% and 0.62 respectively. The performance of the models on independent dataset is also reasonable. Based on the above study, we have developed a user-friendly web server named "AntiAngioPred" for the prediction of anti-angiogenic peptides. AntiAngioPred web server is freely accessible at http://clri.res.in/subramanian/tools/antiangiopred/index.html (mirror site: http://crdd.osdd.net/raghava/antiangiopred/).

DNA damage response and anti-apoptotic proteins predict radiosensitization efficacy of HDAC inhibitors SAHA and LBH589 in patient-derived glioblastoma cells.

HDAC inhibitors have radiosensitizing effects in established cancer cell lines. This study was conducted to compare the efficacy of SAHA, LBH589, Valproic Acid (VPA), MS275 and Scriptaid in the patient-derived glioblastoma model. In more detail, SAHA and LBH589 were evaluated to determine predictors of response. Acetylated-histone-H3, γH2AX/53BP1, (p)Chek2/ATM, Bcl-2/Bcl-XL, p21(CIP1/WAF1) and caspase-3/7 were studied in relation to response. SAHA sensitized 50% of cultures, LBH589 45%, VPA and Scriptaid 40% and MS275 60%. Differences after treatment with SAHA/RTx or LBH589/RTx in a sensitive and resistant culture were increased acetylated-H3, caspase-3/7 and prolonged DNA damage repair γH2AX/53BP1 foci. pChek2 was found to be associated with both SAHA/RTx and LBH589/RTx response with a positive predictive value (PPV) of 90%. Bcl-XL had a PPV of 100% for LBH589/RTx response. Incubation with HDACi 24 and 48 hours pre-RTx resulted in the best efficacy of combination treatment. In conclusion a subset of patient-derived glioblastoma cultures were sensitive to HDACi/RTx. For SAHA and LBH589 responses were strongly associated with pChek2 and Bcl-XL, which warrant further clinical exploration. Additional information on responsiveness was obtained by DNA damage response markers and apoptosis related proteins.

The Bcl-2 inhibitor Obatoclax overcomes resistance to histone deacetylase inhibitors SAHA and LBH589 as radiosensitizers in patient-derived glioblastoma stem-like cells.

Glioblastoma has shown resistance to histone deacetylase inhibitors (HDACi) as radiosensitizers in cultures with Bcl-XL over-expression. We study the efficacy of SAHA/RTx and LBH589/RTx when manipulating Bcl-2 family proteins using the Bcl-2 inhibitor Obatoclax in patient-derived glioblastoma stem-like cell (GSC) cultures. GSC cultures in general have a deletion in phosphatase and tensin homolog (PTEN). Synergy was determined by the Chou Talalay method. The effects on apoptosis and autophagy were studied by measuring caspase-3/7, Bcl-XL, Mcl-1 and LC3BI/II proteins. The relation between treatment response and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status, recurrence and gene expression levels of the tumors were studied. Obatoclax synergized with SAHA and LBH589 and sensitized cells to HDACi/RTx. Over 50% of GSC cultures were responsive to Obatoclax with either single agent. Combined with HDACi/RTx treatment, Obatoclax increased caspase-3/7 and inhibited Bcl-2 family proteins Bcl-XL and Mcl-1 more effectively than other treatments. Genes predictive for treatment response were identified, including the F-box/WD repeat-containing protein-7, which was previously related to Bcl-2 inhibition and HDACi sensitivity. We emphasize the functional relation between Bcl-2 proteins and radiosensitization by HDACi and provide a target for increasing responsiveness in glioblastoma by using the Bcl-2 inhibitor Obatoclax.

Redesigning of anti-c-Met single chain Fv antibody for the cytoplasmic folding and its structural analysis.

Typically, single chain Fv antibodies are unable to fold properly under a reducing cytoplasm because of the reduction of disulfide bonds. The inability to fold limits both the production of the functional scFvs and their targeting against antigens, which are generally executed in a reducing cytoplasm. In this study, the target scFv CDR was grafted with stable human consensus framework sequences, which enabled the generation of a foldable scFv in a reducing cytoplasm of Escherichia coli. Additionally, the structural features affecting the folding efficiency of the engineered scFv were identified by analyzing the predicted structure. An anti-c-Met scFv, which was a cytoplasmic non-foldable protein, was redesigned as the model system. This study confirmed that the engineered anti-c-Met scFv was folded into its native form in the cytoplasm of E. coli BL21(DE3) without a significant loss in the specific binding activity against c-Met antigen. The structures of the wild-type anti-c-Met scFv and the engineered scFv were predicted using homology modeling. A comparative analysis based on the sequence and structure showed that the hydrophobicity of 12 solvent exposed residues decreased, and two newly formed salt bridges might have improved the folding efficiency of the engineered scFv under the reducing condition.